Surface plasmon resonance biosensor for measurement of anti-glycolipid antibody levels in neuropathy

ABSTRACT

The present invention provides a method of detecting human antibodies in a sera solution. The invention also provides a method of quantitating anti-glycolipid antibody levels in solutions. The invention provides a method of diagnosing disease states, including neurological diseases, by quantitating a subject&#39;s antibody levels.

BACKGROUND

[0001] Elevated titers of anti-GM1 ganglioside antibodies (anti-GM1)areassociated with multifocal motor neuropathy, lower motor neuron diseasesyndromes, and with the acute motor axonal neuropathy variant of theGuillian-Barré syndrome.¹⁻⁵ Assays for anti-GM1 antibodies are routinelyused in clinical practice to aid in the evaluation of patients suspectedof having these syndromes.

[0002] Anti-GM1 antibodies are typically measured using the ELISAsystem, in which increasing serum dilutions are tested for binding toGM1-coated microwells.⁶ However, the assay takes several days toperform, is costly, and is done at non-physiologic conditions oftemperature and antibody concentration. Issues of methodology have alsolimited the usefulness of the technique.⁷⁻¹⁰ There is a need for afaster and more reliable test to detect and measure anti-GM1 antibodies.

[0003] Here we disclose a novel method for the rapid detection andquantitation of serum antibodies in serum. In the invention disclosedhere we present data showing quantitation of serum anti-gangliosideantibodies in serum. The system utilizes a surface plasmonresonance-based optical biosensor. Gangliosides are immobilized on thesurface of a sensor chip comprised of a carboxymethyl dextran layerlinked to a thin gold film coated on a glass slide. The sensor chip isbrought in contact with a flow cell carrying the analyte. Applying thephenomenon of surface plasmon resonance, possible interactions betweenthe analyte in the flow cell and the gangliosides, leading to changes inthe refractive index at the surface of the chip, can be followed.¹¹⁻³²The binding of anti-GM1 antibodies in a sample to immobilized GM1 can beobserved in real time without the use of secondary labels. The inventiondisclosed here reveals a method of diagnosing human diseases bydetection and quantitation of antibodies in human sera.

SUMMARY OF THE INVENTION

[0004] The present invention provides a method of detecting antibodiesin a solution comprising:

[0005] a) contacting the solution with an antigen-coated surface of asensor chip under conditions that permit anti-antigen antibodies to bindto the antigen coating;

[0006] b) detecting the change in surface plasmon resonance signal ofthe sensor chip resulting from the anti-antigen antibodies binding tothe antigen coating.

[0007] The present invention provides a method of detecting antibodiesas described hereinabove, wherein the antigen is a glycolipid.

[0008] The present invention provides a method of detecting antibodiesas described hereinabove wherein the anti-antigen antibodies areanti-glycolipid antibodies.

[0009] The present invention provides a method of detecting antibodiesas described hereinabove wherein the antigen is a ganglioside and theanti-antigen antibodies are anti-ganglioside antibodies.

[0010] The present invention provides a method of detectinganti-glycolipid antibodies in a solution wherein the solution containsanti-glycolipid antibodies that bind to the glycolipid-coated surface ofthe sensor chip and alter the surface plasmon resonance.

[0011] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the glycolipid is a ganglioside and wherein a control surfaceplasmon resonance value is subtracted from the surface plasmon resonancesignal.

[0012] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the control surface plasmon resonance value comprises the signaldetected from the surface of the sensor chip coated with a controlantigen, wherein the chip, is also alternatively exposed to the solutionbeing evaluated for anti-glycolipid antibodies. The present inventionalso provides a method wherein the control antigen is ganglioside GM2.The present invention also provides a method wherein the anti-antigenantibody is anti-ganglioside GM1 antibody.

[0013] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the surface plasmon resonance signal is detected using anoptical detector.

[0014] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the solution is human blood or a derivative of human blood.

[0015] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the solution is human sera.

[0016] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the anti-glycolipid antibody is an anti-glycolipidImmunoglobulin G.

[0017] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the anti-glycolipid antibody is an anti-glycolipidImmunoglobulin M.

[0018] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the anti-glycolipid antibody is an anti-ganglioside antibody.

[0019] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the anti-glycolipid antibody is human.

[0020] The present invention provides a method of determining theanti-glycolipid antibody isotype present in the solution comprising themethods described hereinabove, wherein the tested solution is washedfrom the surface of the sensor chip and a second solution containing asecondary antibody is introduced to the surface.

[0021] The present invention provides a method of increasing the opticalsignal size of the methods described hereinabove, comprising washing thetested solution from the surface of the sensor chip and applying thesecond solution containing the secondary antibody to the surface.

[0022] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the secondary antibody is an anti-Immunoglobulin G.

[0023] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the secondary antibody is an anti-Immunoglobulin M.

[0024] The present invention provides the use of the methods describedhereinabove in diagnosing a disease in a subject.

[0025] The present invention provides the use of the methods describedhereinabove in diagnosing a neurological disease in a subject.

[0026] The present invention provides the use of the methods describedhereinabove in quantitating levels of anti-antigen antibodies in asubject.

[0027] The present .invention provides the use of the method describedhereinabove in evaluating a neuropathic disease in a subject, whereinthe disease is Guillian-Barré syndrome, motor neuropathy, peripheralneuropathy or autoimmune neuropathy.

BRIEF DESCRIPTION OF THE FIGURES

[0028]FIG. 1. Schematic representation of a surface plasmon resonancebiosensor, which is based on the phenomenon of total internal reflectionof light. As anti-GM1 antibodies bind the ganglioside-coated sensorchip, the resulting changes in the refractive index of the solution incontact with the chip can be detected by monitoring the change in SPRangle. FIG. 2A and 2B. Presence of anti-GM1 antibodies in serum isdetected and visualized in the form of sensorgrams. (A) Two sensorgramsresult from the interaction of sample with the GM1- and GM2-coatedsurfaces of the sensor chip. (B) Subtraction of response for the controlflow cell from response for GM1-coated flow cell results in a correctedsensorgram indicating net binding of anti-GM1 antibodies. Finalmeasurement of response difference is taken at 20 seconds after the endof injection. S=Start of injection, E=End of injection.

[0029]FIG. 3. Use of a secondary antibody for signal enhancement. (A)Injection of a 1:100,000 dilution of an MMN serum sample with anti-GM1antibodies. (B) Introduction of a 0.5 mg/mL solution of goat anti-humanIgM following sample injection, enhanced the signal nearly six fold.

[0030]FIG. 4A and 4B. Use of secondary antibodies for identification ofthe anti-GM1 autoantibody isotype. (A) Analysis of a serum sample froman MMN patient with anti-IgM and anti-IgG antibodies indicated that theantibodies in the serum are of the IgM class. (B) Similar analysis of asample from a GBS patient indicated the presence of IgG classantibodies.

[0031]FIG. 5. Corrected sensorgrams for a serially diluted serum sample(1:5 to 1:800) from a representative MMN patient with anti-GM1antibodies.

[0032]FIG. 6. Linearity of the assay was examined with serial dilutionsof a serum sample in HBS-N buffer containing 1 mg/mL carboxymethyldextran.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The following definitions are presented as an aid inunderstanding this invention:

[0034] ALS—Amyotrophic Lateral Sclerosis;

[0035] CIDP—Chronic Inflammatory Demyelinating Polyneuropathy;

[0036] ELISA—Enzyme-linked Immunosorbent Assay;

[0037] GBS—Guillian-Barré syndrome;

[0038] GM1—Ganglioside G_(M1);

[0039] GM2—Ganglioside G_(M2);

[0040] IgG—Immunoglobulin G;

[0041] IgM—Immunoglobulin M;

[0042] MAG—Myelin Associated Glycoprotein;

[0043] MMN—Multifocal Motor Neuropathy;

[0044] SPR—Surface Plasmon Resonance.

[0045] Having due regard to the preceding definitions, the presentinvention provides a method of detecting antibodies in a solutioncomprising:

[0046] a) contacting the solution with an antigen-coated surface of asensor chip under conditions that permit anti-antigen antibodies to bindto the antigen coating;

[0047] b) detecting the change in surface plasmon resonance signal ofthe sensor chip resulting from the anti-antigen antibodies binding tothe antigen coating.

[0048] In one embodiment the antigen coated surface is aglycolipid-coated surface. In one embodiment the antigen coated surfaceis a protein coated surface. In one embodiment the surface coated withganglioside GM1. In another embodiment the glycolipid-coated surface iscoated with ganglioside GM2. In one embodiment the solution being testedis human sera. In another embodiment the antibodies being detected arehuman antibodies. In one embodiment the surface plasmon resonance signalis detected by an optical detection apparatus. In one embodiment thebinding of antibodies to the antigen surface causes a change in thesurface plasmon resonance signal. In one embodiment the antigen iscoated onto the surface of the chip using any common means including,but not limited to, covalent linking and conjugation.

[0049] The present invention provides a method of detecting antibodiesas described hereinabove, wherein the antigen is a glycolipid.

[0050] The present invention provides a method of detecting antibodiesas described hereinabove wherein the anti-antigen antibodies areanti-glycolipid antibodies.

[0051] The present invention provides a method of detecting antibodiesas described hereinabove wherein the antigen is a ganglioside and theanti-antigen antibodies are anti-ganglioside antibodies.

[0052] The present invention provides a method of detectinganti-glycolipid antibodies in a solution wherein the solution containsanti-glycolipid antibodies that bind to the glycolipid-coated surface ofthe sensor chip and alter the surface plasmon resonance. In oneembodiment the anti-glycolipid antibodies are anti-ganglioside GM1antibodies. In one embodiment the glycolipid-coated surface is aganglioside GM1-coated surface.

[0053] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the glycolipid is a ganglioside and wherein a control surfaceplasmon resonance value is subtracted from the surface plasmon resonancesignal. In one embodiment the surface plasmon resonance signal isdetected by an optical detection apparatus. In one embodiment thecontrol surface plasmon resonance value is measured when the surface ofthe sensor chip is coated with a control antigen.

[0054] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the control surface plasmon resonance value comprises the signaldetected from the surface of the sensor chip coated with a controlantigen, wherein the chip is also alternatively exposed to the solutionbeing evaluated for anti-glycolipid antibodies. The present inventionalso provides a method wherein the control antigen is ganglioside GM2.The present invention also provides a method wherein the anti-antigenantibody is anti-ganglioside GM1 antibody.

[0055] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the surface plasmon resonance signal is binding ofanti-glycolipid antibodies to the glycolipid-coated surface of a sensorchip alters the refractive index of the surface and thus alters theoptical signal detected.

[0056] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the solution is human blood or a derivative of human blood.Derivative of human blood includes, but is not limited to; centrifugedblood products and sera.

[0057] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the solution is human sera. Human sera includes sera derivativesor modified sera as well as plain sera.

[0058] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the anti-glycolipid antibody is an anti-glycolipidImmunoglobulin G.

[0059] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the anti-glycolipid antibody is an anti-glycolipidImmunoglobulin M.

[0060] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the anti-glycolipid antibody is an anti-ganglioside antibody. Inone embodiment the anti-glycolipid antibody is an anti-ganglioside GM1antibody.

[0061] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the anti-glycolipid antibody is human. In one embodiment theanti-glycolipid antibody is a human anti-ganglioside GM1 antibody.

[0062] The present invention provides a method of determining theanti-glycolipid antibody isotype present in the solution comprising themethods described hereinabove, wherein the tested solution is washedfrom the surface of the sensor chip and a second solution containing asecondary antibody is introduced to the surface. In one embodiment thesensor chip surface is spatially separated into two distinct areas, onecoated with the test antigen and the other coated with the controlantigen. In one embodiment the antibody being quantitated binds to thetest antigen. Quantitating the test antibody level means comparing thechange in surface plasmon resonance signal seen when the test solutionis introduced to the test antigen coated surface to the change incontrol surface plasmon resonance signal seen when the test solution isintroduced to the control antigen coated surface. The test antibody isthe antibody being tested for by the method, i.e. the antibody that canbind to the antigen-coated surface of the sensor chip.

[0063] The present invention provides a method of increasing the opticalsignal size of the methods described hereinabove, comprising washing thetested solution from the surface of the sensor chip and applying thesecond solution containing the secondary antibody to the surface. In oneembodiment the secondary antibody binds to test antibody which in turnis bound to the antigen-coated surface of the sensor chip. In oneembodiment the secondary antibody bound to the test antibody which inturn is bound to the antigen-coated surface of the sensor chip providesa bigger change in surface plasmon resonance signal than the testantibody alone bound to the antigen-coated surface of the sensor chip.

[0064] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the secondary antibody is an anti-Immunoglobulin G.

[0065] The present invention provides a method of detectinganti-glycolipid antibodies in a solution as described hereinabove,wherein the secondary antibody is an anti-Immunoglobulin M.

[0066] The present invention provides the use of the methods describedhereinabove in diagnosing a disease in a subject. Diagnosing a diseasemeans identifying that disease state in a subject. In one embodiment thedisease is diagnosed by quantitating the levels of an antibody in asubject. In a further embodiment the antibody is a characteristic of thedisease state. In another embodiment an antibody level in the subject isa characteristic of the disease state.

[0067] The present invention provides the use of the methods describedhereinabove in diagnosing a neurological disease in a subject.

[0068] The present invention provides the use of the methods describedhereinabove in quantitating levels of anti-antigen antibodies in asubject. In one embodiment the quantitation of levels of anti-antigenantibodies in a subject is used for assessing the success of treatingthe disease in a subject.

[0069] The present invention provides the use of the method describedhereinabove in evaluating a neuropathic disease in a subject, whereinthe disease is Guillian-Barré syndrome, motor neuropathy, peripheralneuropathy or autoimmune neuropathy. In one embodiment the presence ofhuman anti-glycolipid antibodies in the test solution is used as anindicator in diagnosing the diseases mentioned hereinabove.

Experimental Results

[0070] Sera from a total of 26 individuals were examined for anti-GM1antibodies by surface plasmon resonance biosensor assay and ELISA. Ofthe eight sera examined from MMN patients, five tested positive foranti-GM1 antibodies by the SPR assay, while four were positive by ELISA.Of the two sera examined from GBS patients, one was positive foranti-GM1 antibodies by both assays. All sera from patients with CIDP,ALS, demyelinating neuropathy associated with anti-MAG antibodies, andMiller Fisher variant of GBS, as well as those from normal subjectstested negative for anti-GM1 antibodies by SPR biosensor assay and ELISA(table 1).

[0071] Signal enhancement was studied by analysis of a sample withanti-GM1 antibody titer of 51,200, as determined by ELISA. Detection ofanti-GM1 antibody in the sample was possible with at least 100,000 folddilution using the biosensor. Signal due to interaction of antibody withthe antigen-coated sensor chip was enhanced by increasing sample contacttime with the chip, either by using a higher sample volume or bydecreasing the flow rate. A dramatic enhancement of sensorgram response,however, was possible with the aid of a secondary antibody. While a100,000 fold dilution of the sample yielded a response of 11.2±0.5 RU,injection of a 0.5 mg/mL solution of goat anti-human IgM (25 μL at 10μL/min) following sample injection (100 μL at 10 μL/min), increased theresponse by 55.9±2.1 RU (FIG. 3).

[0072] Secondary antibodies were also utilized as a tool for identifyingthe anti-ganglioside immunoglobulin isotype. To demonstrate their use,0.1 mg/mL solutions of goat anti-human IgM or anti-human IgG wereinjected (25 μL at 25 μL/min), following injections of 1:40 dilution ofa serum sample from a representative patient (no. 2): with MMN (FIG.4A). As expected, only anti-IgM antibodies demonstrated binding to thesensor chip surface, resulting in an increase in response, andindicating the presence of IgM anti-GM1 antibodies in sample. Thisprocedure was repeated for a serum from a patient with Guillian-Barrésyndrome and antibodies against GM1. Only anti-IgG antibodies showedbinding, demonstrating the presence of IgG class anti-GM1 antibodies inthis sample (FIG. 4B).

[0073] Linearity of the assay was tested by analyzing serial dilutions(1:5 to 1:800) of a representative serum sample (no. 2) from a patientwith MMN (FIG. 5). The plot of response as a function of dilutiondemonstrated linearity at 1:50 and higher dilutions (r²=0.9941) (FIG.6). At higher concentrations, the curve became logarithmic, such thatthe plot of response as a function of log dilution yielded linearitybetween 1:5 and 1:100 dilutions (r²=0.9949). Serial dilution of othersera showed similar results.

[0074] Table 1. Analysis of patient sera with SPR biosensor assay andELSIA Number of serum Number positive Number positive by samples byELISA SPR biosensor assay MMN 8 4 3 ClDP 5 0 0 ALS 3 0 0 Anti-MAGNeuropathy 3 0 0 GBS 2 1 1 Normal 5 0 0

Materials and Methods

[0075] Serum Samples. Serum samples were obtained from 21 patients:eight with multifocal motor neuropathy (MMN), five with chronicinflammatory demyelinating polyneuropathy (CIDP), three with amyotrophiclateral sclerosis (ALS) three with demyelinating neuropathy associatedwith anti-myelin-associated glycoprotein (anti-MAG) antibodies, and twowith Guillian-Barré syndrome (GBS). One of the GBS patients wasdiagnosed with the Miller Fisher variant, with antibodies against GQ1bganglioside. Criteria for patient classification have previously beendescribed.^(2, 13-15) In addition, sera from five normal subjects wereevaluated as controls. All patient sera were stored at −20 ° C.

[0076] Instrument. Binding of antibodies to gangliosides was measuredwith a BIAcore X instrument (Biacore, Uppsala, Sweden). The system wascomprised of a detector, a sensor chip for ligand immobilization, and asample delivery unit. It utilized surface plasmon resonance, based onthe optical phenomenon of total internal reflection of light, to monitorchanges in refractive index at the surface of the chip, which could bevisualized in the form of sensorgrams (FIG. 1).

[0077] Sensor Chip. A B1 Pioneer sensor chip (Biacore, Uppsala, Sweden)was used in the assay. The chip was composed of a glass slide with agold film covalently linked-to a methyl dextran layer containing a lowdegree of carboxylation. When the microfluidic cartridge of theinstrument was docked against the sensor chip, two flow cells wereformed, each of which could be coated with a different antigen.

[0078] Immobilization of Gangliosides. Gangliosides GM1 and GM2 (SigmaChemicals, St. Louis, Mo.) were used as active and control antigensrespectively in the immunoassay. The ganglioside GM2 was chosen ascontrol because it is one of the least reactive gangliosides andreported cases of the presence of anti-GM2 antibodies in neuropathypatients have been fewer than those for other commercially availablegangliosides. Immobilization was performed by adsorption, with thesensor chip in the instrument. A 250 μg/mL solution of GM1 was preparedby combining 20 μL of a 1 mg/mL stock solution of the ganglioside inethanol with 60 μL of HBS-N buffer [10 mMN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) buffer(pH 7.4), containing 150 mM NaCl]. Immobilization of the ganglioside wasinitiated by introduction of 45 μL of this solution into one of the wasimmobilized on the second flow cell in the same manner. The sensor chipwas incubated at 4° C. for 48 hours, allowing the immobilized moleculesto equilibrate, after which the flow cells were coated a second time asbefore with GM1 and GM2. Approximately 200 RU of each ganglioside wascoated onto the flow cells.

[0079] SPR Analysis of Sera. All specimens were diluted 1:10 with HBS-Nbuffer containing 1 mg/mL carboxymethyl dextran (Fluka, Milwaukee,Wis.). The carboxymethyl dextran was used to reduce nonspecific binding.Samples were injected in triplicates at 25 μL/min for 1 minute usingmultichannel detection. The samples were exposed to both active andcontrol surfaces. Interaction of sample with each of the twoganglioside-coated surfaces of the sensor chip was monitored as aseparate sensorgram. The response was measured in resonance units (RU).Presence of anti-GM1 antibodies in samples was verified by subtractionof the GM2 surface sensorgram from the GM1 surface sensorgram (FIG. 2).

[0080] Positive binding of antibodies to the GM1-coated surfaceindicated the presence of significant levels of anti-GM1 antibodies inthe serum. Negative binding was considered to indicate the absence ofdetectable quantities of anti-GM1 antibodies. Final measurements weretaken at 20 seconds after the end of each injection. After eachmeasurement, the surface was regenerated with one to three 13 μLinjections of 5 to 20 mM NaOH at 25 μL/min, depending on the level ofantibody binding. The existence of a linear range with respect toresponse and antibody concentration was investigated by duplicateanalysis of serially diluted samples with high levels of anti-GM1antibodies.

[0081] Possibility of immunoglobulin class identification and signalenhancement with a secondary antibody was investigated using goatanti-human IgG and goat anti-human IgM. antibodies (Sigma Chemicals, St.Louis, Mo.). Sample injection was followed by injection of a 0.5 or 0.1mg/mL solution of the secondary antibody in HBS-N buffer containing 1mg/mL carboxymethyl dextran.

[0082] Enzyme-Linked Immunosorbent Assay (ELISA). The presence ofanti-GM1 antibodies in sera was also measured by the enzyme-linkedimmunosorbent assay, following previously described procedure,³ withsome modification. Wells in 96-well round-bottom polystyrene microtiterplates (Becton Dickinson, Franklin Lakes, N.J.) were coated with 0.5 ?gof GM1 in 100 μL of methanol. Wells to which only methanol was addedwere used as controls. After the evaporation of methanol, all wells wereblocked by incubation with 300 μL of 1% bovine serum albumin (BSA) in 10mM phosphate-buffered saline (150 mM NaCl, pH 7.4) (PBS) for 4 hours at4° C. Wells were washed with the BSA/PBS solution, and 100 μL of serumserially diluted in the BSA/PBS solution was added to the wells. Theplates were incubated at 4° C. overnight. Plates were washed as before,followed by the addition of 100 μL of peroxidase-conjugated goatanti-human IgM or IgG secondary antibody (ICN Biomedicals, Costa Mesa,Calif.) after dilution in BSA/PBS solution (a final concentration of2.14 μg/mL) to each well, and incubation for 2 hours at 4° C. Plateswere washed again and 100 μL of developing solution comprised of 27 mMcitric acid, 50 mM Na₂HPO₄, 5.5 mM o-phenylenediamine, and 0.01% H₂O₂(pH 5-5.5) was added to each well. The plates were incubated at roomtemperature for 30 minutes before measuring absorbance at 450 nm. Thetiter for each specimen was assigned as the highest dilution at whichthe absorbance reading was 0.1 units greater than in the correspondingcontrol wells. Sera with titers of 800 or below were considered to benegative for the presence of clinically significant amounts of anti-GM1antibodies, as such titers are also seen in normal subjects.

Discussion

[0083] This invention provides an SPR-based biosensor for the detectionand measurement of antibodies in human serum. Presence of anti-GM1antibodies was detected and visualized in the form of sensorgrams, usingsurface plasmon resonance. Interaction and specific binding of serumantibodies to the GM1-coated sensor surface were characterized by apositive response after subtraction of control sensorgram from the GM1sensorgram. A negative response was indicative of absence of anti-GM1antibodies in the sample. The biosensor assay was also capable ofidentifying antibody isotype by following sample injection with theintroduction of a secondary antibody.

[0084] Assay reproducibility was evaluated by triplicate analysis of allsamples. The assay exhibited high reproducibility, with standarddeviations of triplicate results less than 7.1% of the mean values.Results of sample positivity or negativity were not altered in any ofthe repeat measurements. With the regeneration conditions used in thisstudy, the coated sensor chip surface was stable for up to approximately90 injections.

[0085] The biosensor immunoassay appeared to be specific for MMN and GBSpatients with antibodies against GM1, as none of the other patients ornormal subjects exhibited positive results. Three specimens withelevated levels of serum IgM and increased titers of anti-MAGantibodies, as well as a specimen from a patient with the Miller Fishervariant of GBS and increased titer of anti-GQ1b antibodies, testednegative for reactivity to GM1 with the biosensor assay.

[0086] With regard to sensitivity, the new assay exhibited similarresults when compared to the ELISA system. It gave positive results inall four of the eight patients with MMN and elevated anti-GM1 antibodiesas determined by ELISA, with titers ranging between 1,600 and 51,200.One other patient with MMN was positive by the new assay but negative byELISA, with a titer of 800. The three remaining patients with MMN werenegative for anti-GM1 antibodies by both the SPR-biosensor and ELISAsystems. Furthermore, the biosensor assay was capable of distinguishingbetween sera from patients 2 and 7 with equal antibody titers. Althoughboth had a titer of 6,400, as determined by ELISA, the SPR assay yieldedresponses of 425.9 RU and 243.5 RU. This may be due to the fact that incontrast with the ELISA system, where information is provided in theform of antibody titers which are the result of analysis of seriallydiluted samples, the SPR biosensor assay yields data directlyrepresentative of serum antibody concentration and affinity, in equallydiluted sera. To be able to differentiate between the above two samplesby the ELISA system with accuracy equivalent to that of the biosensorassay, significantly smaller dilutions would be necessary.

[0087] Furthermore, in comparison with ELISA, the biosensor assay wasperformed under more physiologic elements of temperature andconcentration, and the process of incubation was eliminated. Therefore,the antibody-antigen interaction, as analyzed by the biosensor assay, ismore representative of that in the human body than ELISA.

[0088] With the small number of samples analyzed, the assay showed goodspecificity and sensitivity. The new biosensor assay is fast, withsubstantially reduced sample preparation when compared to ELISA. UnlikeELISA, which is tedious and time-consuming, the biosensor assay revealedhere can be completed in minutes and can be used as a rapid screeningprocedure for the presence of anti-GM1 antibodies. It can help inpatient diagnosis as well as in accurate monitoring of patient serumantibody levels in response to treatment. In addition to testing forantibodies to GM1, the SPR biosensor immunoassay can be used indetecting antibody reactivities to other individual gangliosides, organglioside mixtures, by replacing the GM1 coating of the sensor chipwith other antigens.

References

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1. A method of detecting antibodies in a blood or blood derivativesolution comprising: a) contacting the blood or blood derivativesolution with a glycolipid-coated surface of a sensor chip underconditions that permit the antibodies to bind to the glycolipid coating;and b) detecting a change in surface plasmon resonance signal of thesensor chip resulting from the antibodies binding to the glycolipidcoating.
 2. (Currently Canceled).
 3. The method of claim 1, wherein theantibodies are anti-glycolipid antibodies.
 4. (Currently Canceled). 5.The method of claim 3, wherein the solution contains anti-glycolipidantibodies that bind to the glycolipid-coated surface of the sensor chipand alter the surface plasmon resonance.
 6. The method of claim 1,wherein a control surface plasmon resonance value is subtracted from thesurface plasmon resonance signal.
 7. The method of claim 6, wherein thecontrol surface plasmon resonance value comprises the signal detectedfrom the surface of the sensor chip coated with a selected controlglycolipid, wherein the chip is also alternatively exposed to thesolution being evaluated for anti-glycolipid antibodies.
 8. (CurrentlyCanceled).
 9. (Currently Canceled).
 10. (Currently Canceled).
 11. Themethod of claim 1, wherein the sensor chip comprises a glass slidecoated with a gold film covalently linked to a methyl dextran layer. 12.The method of claim 1, wherein the surface plasmon resonance signal isdetected using an optical detector.
 13. The method of claim 1, whereinthe solution is human blood or a derivative of human blood.
 14. Themethod of claim 13, wherein the derivative of human blood is human sera.15. The method of claim 3, wherein the anti-glycolipid antibody is anImmunoglobulin G.
 16. The method of claim 3, wherein the anti-glycolipidantibody is an Immunoglobulin M.
 17. (Currently Canceled).
 18. Themethod of claim 17, wherein the antibody is a human antibody.
 19. Themethod of claim 3, for determining an isotype for the anti-glycolipidantibody present in the solution further comprising washing the testsolution from the surface of the sensor chip after step (b); applying asecond solution containing a secondary isotype-specific antibody to thesurface; and detecting a change in surface plasmon resonance signal ofthe sensor chip resulting from the secondary isotype-specific antibodybinding to the anti-glycolipid antibody thereby determining the isotypeof the anti-glycolipid antibody.
 20. The method of claim 1, comprisingwashing the blood or blood derivative solution solution from the surfaceof the sensor chip and applying a second solution containing a secondaryantibody to the surface to increase the surface plasmon resonancesignal.
 21. The method of claim 19, wherein the secondary antibody is ananti-Immunoglobulin G.
 22. The method of claim 19, wherein the secondaryantibody is an anti-Immunoglobulin M.
 23. A method of diagnosing adisease in a subject which comprises obtaining a blood or bloodderivative solution from the subject and detecting antibodies thereinaccording to the method of claim 1 wherein the antibody is acharacteristic of the disease state and detecting the antibody in theblood or blood derivative solution from the subject identifies thedisease in the subject.
 24. A method of quantitating levels ofantibodies in a subject which comprises obtaining a blood or bloodderivative solution from the subject and detecting antibodies thereinaccording to the method of claim 7 and quantitating the level ofantibodies by comparing any change in surface plasmon resonance signalof the sensor chip when a control solution is introduced to theglycolipid coated surface and wherein an antibody level ischaracteristic of the disease.
 25. The method of claim 23, wherein thedisease is a neurological disease.
 26. The method of claim 25, whereinthe neurological disease is Guillian-Barré syndrome, motor neuropathy,peripheral neuropathy or an autoimmune neuropathy.